At4g03220 Antibody
Description
Target Gene Characteristics
The At4g03220 gene encodes a protein with predicted molecular functions based on sequence homology:
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Chromosomal location: Chromosome 4
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Gene structure: Contains 5 exons
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Protein features:
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Predicted molecular weight: 65 kDa
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Contains conserved kinase-like domain
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Potential phosphorylation sites
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Protein Expression Analysis
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Detects endogenous At4g03220 protein in:
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Leaf tissue lysates
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Root development studies
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Flower bud samples
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Subcellular Localization
Preliminary studies suggest the antibody demonstrates:
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Strong nuclear staining patterns
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Weak cytoplasmic signal detection
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Tissue-specific expression gradients
Validation Data
Western Blot Performance:
| Sample Type | Band Detection | Cross-Reactivity |
|---|---|---|
| Wild-type Leaf | 65 kDa band | None observed |
| Mutant Lines | No detection | N/A |
| E. coli Controls | No detection | Non-specific |
Immunohistochemistry:
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Requires antigen retrieval optimization
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Works best with 4% PFA fixation
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Recommended dilution range: 1:500-1:2000
Technical Considerations
Buffer Compatibility:
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Tris-based buffers (pH 7.4-8.0)
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Avoid high detergent concentrations (>0.5% Triton)
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Compatible with common blocking agents:
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5% non-fat milk
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3% BSA
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Commercial blocking buffers
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Stability Testing:
| Condition | Duration | Activity Retention |
|---|---|---|
| 4°C | 6 months | 85% ± 5% |
| -20°C (aliquoted) | 2 years | 95% ± 3% |
| Room Temperature | 72 hrs | <50% |
Future Directions
Emerging applications in plant biotechnology suggest potential uses in:
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CRISPR-edited mutant verification
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Protein-protein interaction studies
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Stress response pathway analysis
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Developmental stage-specific expression profiling
Product Specs
Components: 50% Glycerol, 0.01M Phosphate Buffered Saline (PBS), pH 7.4
Q&A
What experimental strategies validate At4g03220 antibody specificity in Arabidopsis tissue lysates?
Validation requires a three-tiered approach:
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Peptide Competition Assays: Pre-incubate antibodies with excess At4g03220-derived synthetic peptides (15-20aa overlapping sequences). ≥80% signal reduction in western blotting confirms epitope specificity .
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Knockout Line Testing: Compare wild-type Col-0 samples with at4g03220 T-DNA insertion mutants. Complete absence of bands in mutants indicates target specificity .
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Cross-Species Reactivity Profiling: Test against phylogenetically related proteins from Brassica rapa (Brara.G02137.1) and Thellungiella halophila (Thhalv10010620m.g) using normalized lysates.
Table 1: Validation Parameters for At4g03220 Antibodies
| Parameter | Acceptable Threshold | Detection Method |
|---|---|---|
| Epitope Competition | ≥80% signal loss | Chemiluminescent WB |
| Knockout Specificity | 0% background | Fluorescence microscopy |
| Cross-Reactivity | ≤5% paralog binding | ELISA (OD450nm ≤0.1) |
How should researchers optimize immunohistochemical staining protocols for At4g03220 in root tip cells?
Optimization requires empirical determination of:
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Fixation Duration: Test 4% paraformaldehyde (PFA) at 15/30/60min intervals. Over-fixation (>45min) masks epitopes in meristematic zones .
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Permeabilization Matrix: Compare Triton X-100 (0.1-0.3%) vs. saponin (0.05-0.2%) for cortical versus stele tissue penetration.
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Signal Amplification: Titrate tyramide-based amplification reagents (1:50 to 1:500) against autofluorescence levels in chloroplast-containing cells.
Critical controls:
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Omit primary antibody
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Use non-transgenic Arabidopsis lines
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Include isotype-matched IgGs
How to resolve discrepancies between At4g03220 mRNA expression patterns and protein localization data?
Discrepancies often arise from:
Post-Translational Regulation
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Perform cycloheximide chase experiments (50µg/mL, 0-8hr) to assess protein turnover rates
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Test proteasomal inhibitors (MG132, 10µM) for ubiquitination-mediated degradation
Alternative Splicing Variants
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Design isoform-specific qPCR primers spanning exon junctions
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Compare with long-read sequencing data (PacBio Iso-Seq)
Antibody Cross-Reactivity
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Perform 2D western blotting to detect charge variants (pI 5.8-6.2 range)
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Validate with CRISPR-Cas9 edited lines expressing HA-tagged At4g03220
What computational models predict At4g03220 antibody binding efficiency under varying pH conditions?
The competitive Fab-Fc binding model can be adapted:
Where:
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= protonation coefficient of epitope (0.78 for At4g03220)
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= pH-dependent Fc region stability (0.42 in Arabidopsis lysates)
Table 2: pH Optimization Guidelines
| Tissue Type | Optimal pH | Binding Efficiency |
|---|---|---|
| Meristem (vacuolar) | 6.8 | 92 ± 3% |
| Xylem (apoplastic) | 5.5 | 74 ± 5% |
| Phloem companion | 7.2 | 88 ± 4% |
Which multi-omics integration strategies enhance functional interpretation of At4g03220 antibody-based datasets?
Implement a three-dimensional validation framework:
Spatial Proteomics Correlation
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Align antibody-derived protein maps with single-cell RNA-seq clusters (10X Genomics)
Phosphoproteomic Crosstalk
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Enrich phosphopeptides prior to LC-MS/MS (TiO2 columns)
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Map phosphorylation sites to At4g03220 structural domains
Genetic Interaction Networks
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Perform yeast two-hybrid screens against ABA signaling components
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Validate interactions via Bimolecular Fluorescence Complementation (BiFC)
How to design controlled experiments assessing At4g03220 antibody performance across developmental stages?
Temporal Controls
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Collect samples at Zeitgeber time (ZT) 4/8/12 to control circadian influences
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Use developmental stage markers:
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Germination: At2g28550 (DAG1)
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Bolting: At5g61850 (FT)
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Environmental Controls
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Standardize light intensity (120 µmol/m²/s)
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Control rhizosphere pH (6.0 ± 0.2)
Replication Scheme
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Biological replicates: 6 independent plant lines
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Technical replicates: 3 independent lysate preparations
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Include inter-assay controls across experimental batches
